Engineered nucleases can be used to effectively manipulate genes in living cells or whole organisms by creating site-specific double-strand breaks at desired locations in the genome (Nat Rev Genet, 2014. 15(5): p. 321-34.). Engineered nucleases, which comprise a DNA-binding domain and a nuclease domain customized for type II restriction enzymes, have a broad spectrum of genome engineering applications in the biotechnology and medical fields as well as various other industries. More recently, a more potent RGEN platform has been developed based on the CRISPR/CAS9 bacterial adaptive immune system.
The sequence that RGEN targets is limited to a protospacer adjacent motif (PAM), which is a DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease. The PAM sequence was not previously reprogrammable in the CRISPR bacterial adaptive immune system. The canonical PAM comprises the sequence 5′-NGG-3′ and is associated with the RGEN derived from the CAS9 nuclease of Streptococcus pyogenes. Hence, the GG motif is a prerequisite for DNA recognition by the RGEN. To expand sequences for use as PAMs, attempts have been made to separate RGENs from different bacterial species with versatile PAMs. In fact, different PAMs have been found to be associated with the CAS9 protein of the bacteria Streptococcus thermophilus (PAM: NNAGAAW) and Neisseria meningitidis (PAM: NNNNGATT), widening the range of selection in determining RGEN target loci.